Center for Clinical Genetics and Genomics

Jerry Vockley, MD, PhD

Positions Held

Director of the Center for Rare Disease Therapy, Children’s Hospital of Pittsburgh

Professor of Pediatrics, University of Pittsburgh School of Medicine

Professor of Human Genetics, University of Pittsburgh Graduate School of Public Health

Cleveland Family Professor of Pediatric Research, University of Pittsburgh

Education

BS in Biology, Carnegie-Mellon University, Pittsburgh, PA

MD, PhD (Genetics) University of Pennsylvania School of Medicine, Philadelphia, PA

Residency in Pediatrics University of Colorado, Denver, CO

Fellowship in Human Genetics, Yale University. School of Medicine, New Haven CT

Research

My research focuses on mitochondrial energy metabolism, branched chain amino acid metabolism, inborn errors of metabolism, and development of novel therapies for inborn errors of metabolism. I also have a strong interest in the genetics of the Plain Communities (Amish and Mennonites) and identification of novel genetic disorders in the general population.

Characterization of a Multifunctional Fatty Acid Oxidation Complex.Disorders of mitochondrial (FAO) are among the most frequent identified through newborn screening in the US. FAO is traditionally viewed as an energy-generating, catabolic pathway but intermediates of this pathway can serve as key substrates for synthesis of other complex lipids. Moreover, mitochondrial energy production requires the functional interaction of three major mitochondrial metabolic pathways for maximum efficiency. Genetic disorders of these pathways are important causes of disease in humans, and clinical findings suggest interruption of one pathway can cause more global dysregulation energy metabolism. We have examined key points of crosstalk between fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) through direct physical measurements. We show that these two pathways exist as an integrated protein complex within mitochondria. We also demonstrate that the FAO mitochondrial trifunctional protein (TFP), which generates NADH+, interacts with the NADH+ binding subunit of complex I of OXPHOS, while the electron transfer flavoprotein Co-Q oxidoreductase (ETFCoQOR) of FAO interacts directly with the coenzyme Q10 binding subunit of complex III. Our findings prove the existence of a multifunctional energy complex within mitochondria and provide an opportunity to better understand the clinical implications of inborn errors of energy metabolism.

VLCAD. More than 100 cases of VLCAD deficiency have been documented in the literature, with three different disease phenotypes. A severe infant-onset form is characterized by acute metabolic decompensation with hypoketotic hypoglycemia, dicarboxylic aciduria, liver dysfunction, and cardiomyopathy. A second form of the disease presents later in infancy or childhood but has a milder phenotype without cardiac involvement. The third form is of adolescent or adult onset and is dominated by muscle dysfunction that is often induced by exercise. Not surprisingly, children with the severe phenotype tended to have null mutations (71% of identified alleles in these patients), whereas patients with the two milder forms of the disease were more likely to have missense mutations (82% and 93% of identified alleles for the milder childhood form and the adult form, respectively). Nevertheless, a few missense mutations were clearly associated with the severe phenotype. Although these data suggested that missense mutations in VLCAD might obviate clinical symptoms due to some degree of residual activity, no correlation was seen between the mutations identified and residual VLCAD activity in fibroblasts. Moreover, the function effects of few of the known VLCAD missense mutations have been directly characterized.

Team researchers have previously used prokaryotic expression systems to express, purify, and characterize the biochemical properties of several ACD enzymes. Several of these have been crystallized and studied by X-ray diffraction, yielding informative three-dimensional models.

The study of VLCAD, however, has been limited due to difficulties with prokaryotic expression. These difficulties may be related, in part, to physical properties that distinguish VLCAD from other ACD family members. Most of the ACDs share a common homotetrameric “dimer of dimers” structure and function in the mitochondrial matrix. In contrast, VLCAD is a homodimer with an extended 180 amino acid C-terminal domain of unknown function. Additionally, VLCAD is associated with the inner mitochondrial membrane, an interaction that has long been postulated without proof to be mediated by the C-terminus.

The team has used its prokaryotic expression system to study six previously missense mutations described in VLCAD-deficient patients (T220M, V243A, R429W, A450P, L462P, and R573W). T220M and V243A are the most frequently reported missense mutations in VLCAD-deficient patients. R429W and R573W are among the few missense mutations believed to result in the severe clinical phenotype. A450P and L462P are located in the C-terminal domain unique to VLCAD and ACAD9. Characterization of purified wild-type, A450P, and L462P VLCAD proteins confirmed the long-held assumption that the C-terminus plays a key role in mitochondrial membrane association. The prokaryotic system developed will greatly facilitate investigation of VLCAD structure and function. Funding for this project is included in the above-referenced grant.

ACD9. Mitochondrial β-oxidation of long-chain fatty acyl-CoAs is well recognized as a primary metabolic pathway for maintenance of energy homeostasis and body temperature. However, it also recycles carbons from many long-chain fatty acids for lipid synthesis. Little is known about the mechanistic role of the latter in the pathogenesis of symptoms in genetic defects of β-oxidation, and its derangement may, in part, explain the features of these disorders, such as neurological dysfunction or acute respiratory distress syndrome, which respond poorly to treatment with alternative energy sources. Very long-chain acyl-CoA dehydrogenase (VLCAD) is the dominant long-chain acyl-CoA dehydrogenase (LCAD) in energy generation in human muscle and heart. In contrast, this study provides evidence that acyl-CoA dehydrogenase 9 (ACAD9) and LCAD more likely function in lipid recycling and synthesis in human brain and lung, respectively, supported by their unique substrate utilization and tissue distribution pattern. Furthermore, the team has identified a new genetic deficiency of LCAD presenting with congenital surfactant deficiency. This disorder represents the first in a α, β-oxidation enzyme primarily involved in lipid recycling or synthesis, revealing a new mechanism of pathogenesis in human disease. Funding for this project by the NIH has been renewed, and a supplement through the economic stimulus grant program was received.

Human ACD10. In the last half of the 20th century, the incidence of type 2 diabetes mellitus (T2DM), previously unrecognized in the Pima Indians, began to rise. Multiple factors were postulated to be responsible including environmental factors, such as diet and resultant obesity, along with a number of genetic determinants. ACAD10 was one of 30 genes further examined after demonstrating a significant signal for diabetes in a genome-wide association study (GWAS). In these studies, a single-nucleotide polymorphism (SNP), rs632650, was found to map within intron 2 of ACAD10. The hypothetical ACAD10 protein is structurally related to the ACAD family of mitochondrial flavoproteins, which consists of nine enzymes that are similar in structure and function as they catalyze the α, β-dehydrogenation of their corresponding acyl-CoA substrates. Seven of these ACADs, LCAD, medium-chain acyl-CoA dehydrogenase (MCAD), short-chain acyl-CoA dehydrogenase (SCAD), isovaleryl-CoA dehydrogenase (IVD), isobutyryl-CoA dehydrogenase, 2-methyl-branched chain acyl-CoA dehydrogenase, and glutamate dehydrogenase, are homotetramers (~400-aa per monomer) and two, VLCAD and ACAD9, are homodimers (~640-aa per monomer). In addition to their usual location in the mitochondrial matrix, some ACADs, including LCAD, MCAD, and SCAD, have been shown to be associated with cytoplasmic GLUT4-containing vesicles where they interact with two dileucine motifs on insulin-regulated aminopeptidase (IRAP). Mutation of the dileucine motif of IRAP (amino acids 55–82) eliminates this interaction. In cells, glucose equilibrium is maintained by the GLUT4 response to insulin. The GLUT4- and IRAP-containing vesicles respond to insulin stimulation by translocation to the cell surface. The dileucine motif in IRAP plays a critical role in regulating GLUT4 trafficking. While these findings connect some of the ACADs with insulin dependent transportation of glucose within cells, the physiologic role of the ACAD proteins in this setting is unclear. Skeletal muscle patterns of fatty acid utilization during fasting conditions have been shown to be associated with obesity-related insulin resistance and altered mitochondrial energy metabolism, including fatty acid oxidation. These abnormalities have also been shown to be present in the context of T2DM. We have recently shown that the pattern of acylcarnitines (ACNs), key metabolic intermediates of fatty acid oxidation, in the blood of obese and T2DM participants fall into two distinct patterns. First, the T2DM and obese participants had a similar accumulation of long-chain ACNs that arise from activity in the initial rounds of β-oxidation, consistent with increased flux at entry into mitochondrial β-oxidation. Diabetic participants also displayed a secondary accumulation of various shorter chain ACNs suggestive of inefficient complete fatty acid oxidation or interactions between β-oxidation and ETC. They also showed an inability to efficiently switch from fat metabolism during insulin clamp, as reflected in their inability to lower their ACNs as effectively as either the lean or obese subjects. In contrast, this pattern was not present in obese adolescents, who instead showed metabolic findings suggestive of upregulation of fatty acid oxidation. To characterize the physiologic role of ACAD10 in intermediary metabolism and its possible link to T2DM, we have characterized an ACAD10 gene trap mouse model. Aging animals become obese on a normal diet and develop insulin-resistant hyperglycemia in response to an intraperitoneal glucose challenge. Tissue and blood ACN profiles are similar to those previously described for adult humans with T2DM. Our findings identify ACAD10 deficiency as new monogenic cause of T2DM in mice, and provide valuable insight into its potential role in the development of T2DM in Pima Indians.

A New Disorder in Sterol Metabolism. Defects in cholesterol synthesis result in a wide variety of symptoms from neonatal lethality to the relatively mild dysmorphic features and developmental delay found in Smith-Lemli-Opitz syndrome (SLOS). The team reports here the identification of mutations in SC4MOL as the cause of a newly recognized autosomal recessive syndrome that includes psoriasiform dermatitis, arthralgias, congenital cataracts, microcephaly, and developmental delay. This gene encodes a sterol-C4-methyl-oxidase, catalyzing demethylation of C4-methylsterols in the cholesterol synthesis pathway. C4-methylsterols are members of meiosis-activating sterols (MAS). They are first found in high concentration in testis and ovary and play roles in meiosis activation. In this study, the team found that MAS affect the cell proliferation in both skin and blood. We also found that inhibition of sterol-C4-methyl-oxidase significantly altered the immune regulation in immunocytes. MAS are ligands of the liver X receptors α and β, which are important in regulating not only lipid transport in epidermis, but also the innate and adaptive immunity. Deficiency of SC4MOL represents a new biochemical defect in the cholesterol synthesis pathway, the clinical spectrum of which remains to be defined.

Characterizing the Burden of Genetic Disease in Old Order Amish. The Old Order Amish communities (Plain People) of North America have altered health risks that stem from unbalanced population sampling of European founders followed by genetic drift in derivative generations. These population effects have resulted in a high prevalence of specific genetic disorders that vary from the general population and from each other. Several characteristics of these communities facilitate genetic analysis. Most isolates keep excellent historical and genealogical records. Due to their sociologic and/or geographic isolation, there is usually little or no migration into the group, and the members of the group exhibit relatively homogeneous lifestyles. Large nuclear families are frequent, which provides adequate numbers of affected and unaffected siblings within a sibship for blood samples. The primary genetic advantage, however, results from the interaction of two overlapping phenomena: the founder effect and inbreeding. Mitochondrial DNA mutations have not previously been reported in any Old Order Amish community. We have recently described an Amish family with the MTTL1 mitochondrial gene mutation m.3243A>G. A second patient with a m.13513G>A (D393N) mutation has also been diagnosed. These mutations classically cause mitochondrial, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. We identified the first mutation in a young woman from the Mercer County Amish community at age 15 years. She had a history of developmental delay, short stature, hearing loss, fatigability, and poor appetite. She presented acutely with vomiting, altered mental status, status epilepticus, and lactic acidosis. Magnetic resonance imaging (MRI) of the brain showed a small, focal, left occipital lobe infarct. She subsequently developed other stroke-like episodes in the left occipital and temporal areas. Molecular testing revealed 74% heteroplasmy in saliva for the MELAS 3243A>G mutation. Several members of the extended maternal pedigree exhibit variable clinical problems including developmental delay, mild hypotonia, hearing loss, renal failure, migraine headaches, adult-onset diabetes mellitus, and recurrent miscarriages, but have never had genetic evaluations. A patient from a second Amish family was diagnosed with MELAS/Leigh overlap syndrome resulting from the mitochondrial mutation m.13513G>A(D393N) in the ND5 subunit of respiratory chain complex I, with blood heteroplasmy level of 2% and urine heteroplasmy level of 43%. The proband was diagnosed at 12 years of age with an acute stroke after a history of developmental delay. His lactate was mildly increased. He has subsequently had recurrent strokes and developed Leigh-like basal ganglia and brainstem lesions with progressive spasticity, dysphagia, and weakness. No siblings were affected, and the mother tested negative for the mutation in blood and urine. A third patient in this Amish community has been diagnosed with an autosomal recessive respiratory chain disorder due to a homozygous deletion in the NDUFAF2 gene, which was in one of the nine areas of homozygosity detected on SNP microarray. He had a history of developmental delay, nystagmus, and hypotonia, and was subsequently admitted to the hospital with a viral illness with progressive respiratory failure. He required intubation, and his MRI revealed Leigh-like lesions. He could not be weaned from the ventilator and three weeks later, his repeat MRI showed worsening of lesions with infarction of cerebellar white matter. He remained unresponsive and fulfilled brain death criteria, ultimately dying after discontinuation of life support.

Amish populations are unique in that they represent genetic bottlenecks dating back to the 18th century, distinguishing them from the European population as a whole, as well genetic drift, which has given rise to variable distributions of pathogenic alleles among North American settlements. Our mitochondrial studies and other clinical encounters lead us to the hypothesis that many unrecognized genetic disorders are present in the Mercer County Amish. This is in keeping with studies conducted by the Clinic for Special Children in Strasburg, Pa., founded and led by Holmes Morton. The clinic is a comprehensive care facility for the Plain People in and around Lancaster County. Morton has extensively characterized this population genetically, including variant analysis through whole-genome and exome sequencing. Not surprisingly, the Old Order Amish demonstrate significant population divergence from the general European population. More surprisingly, genetic variants in the Old Order Amish isolate in Lancaster also differ from those in Big Valley, Pa., and those in Cuyahoga County, Ohio. The Mercer County Amish are among the least genetically characterized Amish communities in the United States, with no catalogue of either genetic disorders or variants seen in the community. We have developed a new program to characterize the genetic variability between Amish Mercer County population and other Amish counties by doing whole-exome and mitochondrial DNA sequencing. This will be crucial to determining the phenotype and frequency of other known and unknown genetic disorders in this population. This project will allow us to characterize load of genetic disease in Old Order Amish of Mercer County and identify disorders that can benefit from early treatment.

A Pig Model of PKU. In this project we are developing a better, clinically relevant animal model (miniature pig) for Phenylketonuria (PKU) in order to understand the biomedical bases and to develop therapeutic approaches, especially for mental retardation, neurological, and neuropsychological features. Using bioinformatics and phylogenetic comparison to human, we initially assembled the entire pig Pah gene encoding a 452 amino acid enzyme, and confirmed high expression of PAH in pig liver and kidney. Furthermore, we have successfully targeted deletions and inversions of the Pah gene using a CRISPR/Cas9 RNA-guided nuclease approach. Our studies over the first 8 months of the NPKUA funding period have utilized our in vitro SCH model system and successfully optimized the genome editing reagents and mutation-detection assays for the pig Pah locus. We transfected CRISPR gRNA/Cas9 vectors into two cell lines and used a deletion-PCR assay with primers located in introns 5 and 6 outside the targeted region on genomic DNA; the intensity of the deletion-breakpoint band with each of the four pairs of gRNAs gives an estimate of the relative efficiency of editing, suggesting that the optimal gRNA reagents were the combination of gRNAs 5-1 + 6-2. DNA sequencing confirmed the presence of locus-specific ~ 1,216-bp deletions in both cell types, clearly generated by repair of two DSBs by NHEJ since the breakpoints have minor heterogeneity. In addition, inversion-PCR assays demonstrated the successful detection of specific bands for the two inversion-breakpoints in each cell line transfected with pairs of CRISPR gRNA/Cas9 vectors. Thus, our strategy for genome editing at the pig Pah locus efficiently induces both deletions and inversions at high frequency. The PKU alleles that we detect in pig models may be deletion or inversion alleles, but in each case exon 6 is lost from the Pah transcriptional unit with the end result being a null mutation with loss of exon 6 as well as a frame-shift after histidine residue #170 and premature termination of protein translation. Working with our Missouri collaborators, the CRISPR gRNAs have been shown to function in vivo in pig pre-implantation embryos and a pregnancy has been obtained from embryo transfers of genome-edited embryos (~ 35% modified alleles). Once we identify minipigs having the PKU genotype, these can be used for breeding and to characterize the biochemical and neurobiological phenotype of pigs with PKU. A porcine PKU-model will allow an understanding of the pathophysiology of PKU, and in the future also for maternal PKU syndrome (MPKUS), including mental retardation, abnormal behavior and neurological features, and will provide a clinically accurate animal model for therapeutic testing for PKU.

Clinical Research. I continue to coordinate a vigorous program in clinical research for the treatment of inborn errors of metabolism. Inborn errors of mitochondrial fatty acid oxidation are among the most frequent now identified through expanded newborn screening by tandem mass spectrometry. I revently formed and lead INFORM (the International Network for Fatty Acid Oxidation Research and Management), which provides a collaborative framework for clinicians and basic scientists to exchange information on disorders of fatty acid oxidation and their global effect on metabolism. We have recently had two medications for fatty acid oxidation disorders developed in my laboratory reach Phase 2 and 3 U.S. Food and Drug Administration trials, including triheptanoin, the first drug in development to treat long chain fatty acid oxidation disorders. Additional diseases under study include lysosomal storage diseases, disorders of sterol metabolism, disorders of the urea cycle, and abnormalities of bone mineralization.